Studies on the movement protein encoded by plant virus genome as a determination factor of host range.

本研究旨在探讨植物病毒基因组编码的运动蛋白作为寄主范围的决定因素。利用苹果褪绿叶斑病病毒(ACLSV)和苹果茎沟病毒(ASGV)，获得了以下结果。结论：1.利用表达载体在大肠杆菌中表达了ACLSV(P-209)基因组ORF2编码的50 kDa运动蛋白，并用于制备抗血清。用抗50 kDa蛋白的抗血清进行免疫印迹分析表明，ACLSV的ORF2蛋白在侵染藜麦的细胞壁和细胞膜部分都有检测到。感染组织中的ORF2蛋白的Mr为52 kDa，大于在大肠杆菌中表达的全长ORF2蛋白(50 KDa)。52 kDa蛋白与碱性磷酸酶孵育后，其表观摩尔浓度降低。从52 kDa到50 kDa，强烈表明ACLSV的ORF2蛋白在受感染的植物组织中发生了光短化。免疫胶体金标记法对感染ACLSV的苦瓜组织切片进行标记，发现ORF2蛋白与细胞壁相关。测定了长距离移动缺陷株ACLSV(MO-5)ORF2蛋白的氨基酸序列。MO-5的ORF2蛋白全长460个氨基酸(Mr 50.4 kDa)，与P-209的同源性为80.8%。免疫印迹分析表明，ORF2蛋白存在于感染的藜麦细胞壁和可溶性成分中。测定了日本梨ASGV(V-3)运动蛋白的氨基酸序列。ASGV分离株的运动蛋白序列比较表明，分离株之间运动蛋白高度保守(94.7-99.7%)。

The present study was undertaken to investigate the movement protein encoded by plant virus genome as a determination factor of host range. Using apple chlorotic leaf spot virus (ACLSV) and apple stem grooving virus (ASGV), following results were obtained.1. A putative movement protin of Mr50kDa encoded by the ORF2 of ACLSV (P-209) genome was expressed in Escherichia coli using an expression vector and wasthern used to produce an antiserum.2. Immunoblot analysis using an antiserum raised against the 50kDa protein showedd that the ORF2 protein of ACLSV was detected in both cell wall and cell membrane fractions prepared from intected Chenopodium quinoatissues.3. The ORF2 protein from infected tissues had a Mr52kDa, larger than that of full-length ORF2 protein (50kDa) expressed in E.coli. Incubation of the 52kDa protein with alkaline phosphatase resulted in a decrease in its apparenet mol. wt. from 52kDa to 50kDa, strongly suggested that the ORF2 protein of ACLSV is phoshorylated in infected plant tissues.4. Immunogold labelling of tissue sections of ACLSV-infected C.quionoaplants showed that the ORF2 protein was found associated with cell wall.5. The amino acid sequence of the ORF2 protein of ACLSV (MO-5), defective strain on long distance movement was determined. The ORF2 protein of MO-5 consistes of 460 amino acis (Mr50.4kDa) and shows 80.8% similarity with that of P-209. Immunoblot analysis indicated that the ORF2 protein was detected in cell wall and soluble fraction prepared from infected C.quinoatissues.6. The amino acid sequence of the movement protein of ASGV (V-3) isolated from Japanese pear was determined. The sequence comparisons of the movement protein among ASGV isolated revealed that the movement proteins were highly conserved among isolated (94.7-99.7%).